Eukaryotic genes are regulated by trans-acting factors that interact with adjacent DNA sequences (cis-acting DNA elements), but no one understands the underlying mechanisms. A clear understanding of how gene are regulated is essential for many areas of biomedicine--cancer, heritable diseases, and development, to name a few. Consequently, work in this area is intense. The fact that each of the approximately 100, 000 genes per mammalian genome has a unique pattern of regulation (by hormones, environmental factors, and development) suggests that there is a complex array of gene regulatory mechanisms. It should, however, be feasible to elucidate these mechanisms by studying a wide variety of genes and comparing their similarities and differences. Such an enormous task will require contributions from many laboratories. The long-range goal of this laboratory is to understand the mechanisms that control tissue-specific expression of specific genes. This proposal uses the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene as a model to accomplish this goal by addressing the mechanisms that allow PEPCK expression in the liver. DNA-mediated transformation studies have revealed that DNA sequences within 2100 bp upstream of the PEPCK gene direct preferential expression of chimeric genes in cells and tissues that normally express the native PEPCK gene. The specific aims of this proposal are directed at fine-mapping and characterizing cis-acting DNA regions ("CTREs") that control tissue-specific expression of the PEPCK gene. This will be accomplished with DNA-mediated transformation of cultured cells (transient assays) and transgenic mice using in vitro mutagenized PEPCK/human growth hormone chimeric genes. In addition, the trans-acting factors that bind the PEPCK/human growth hormone chimeric genes. In addition, the trans- acting factors that bind the PEPCK "CTREs" will be assayed and characterized in nuclear extracts. This will be accomplished by DNA-binding assays such as DNAase I footprinting and electrophoretic mobility shift. Once the PEPCK-specific trans- acting factors and their cognate cis-acting elements have been identified in liver it should be possible to determine how cis- acting elements have been identified in liver it should be possible to determine how PEPCK is regulated in liver and other tissues. This information could then be used to understand the more global problem of how genes in general are developmentally regulated.